Cyclodextrin glucanotransferase and its method of manufacture

ABSTRACT

CGTase enzymatically acts on starch, dextrin, amylopectin, and amylose to produce primarily γ-CD, with quantities of β- and α-CD produced being smaller than the quantity of γ-CD produced, has an optimum pH of 10.5-11.0, an optimum temperature of about 60° C., a stable pH of 6-11, and temperature stability, exhibits residual activity of not less than 90 percent with a 15 minute-treatment at 50° C., is produced by culturing a  Bacillus clarkii  (for example, FERM B-7156), reacts with starch or the like to produce principally γ-cyclodextrin.

This is 371 of PCT/JP01/04310 filed May 23, 2001, which claims priorityto Japan Application 2000-151053 filed May 23, 2000.

FIELD OF THE INVENTION

The present invention relates to cyclodextrin glucanotransferase (EC2.4.1.19, referred to as “CGTase” hereinafter), a method ofmanufacturing the same, and a method of manufacturing cyclodextrin(referred to as “CD” hereinafter) employing the same.

BACKGROUND OF THE INVENTION

CGTase (EC 2.4.1.19) is an enzyme functioning on α-1,4-glucans such asstarches to produce cyclodextrins (CD), which are cyclic α-1,4-glucans,through its intramolecular transfer activity. The degree ofpolymerization of the CDs produced by CGTase is chiefly 6-8, with theseproducts being referred to as α-, β and γ-CD, respectively. In additionto this CD-producing reaction, CGTase catalyzes coupling reactions (thering of the CD is opened and the resulting straight-chainoligosaccharide is transferred to a receptor sugar molecule) anddisproportionation reactions (a straight-chain oligosaccharide istransferred to the receptor sugar molecule) through the intramoleculartransfer reaction. Further, albeit weakly, CGTase also catalyzes thehydrolysis reaction of the α-1,4-glucoside bond.

Since CDs can change chemical and physical properties of variousmolecules by making clathrates therewith, CGTase has achieved a positionas an important enzyme in the food, pharmaceutical and cosmeticindustries. Thus, beginning from the CD synthesis reaction by theBacillus macerans enzyme in 1939 (E. B. Tilden and S. J. Pirt, J. Am.Chem. Soc., 63, 2900-2902, 1939), a large number of studies wereconducted, including a search for bacteria producing CGTase and means ofpurifying the enzyme (Sumio Kitahata, Naoto Tsuyama and Shigetaka Okada,Agr. Biol. Chem., 38 (2), 387-393, 1974; Sumio Kitahata and ShigetakaOkada, Agr. Biol. Chem., 38 (12), 2413-2417, 1974; Sumio Kitahata andShigetaka Okada, J. Jap. Soc. Starch Sci., 29 (1), 13-18, 1982; MichioKubota, Yoshiki Matsuura, Shuzo Sakai and Yukiteru Katsube, DenpunKagaku, 38 (2), 141-146, 1991; Lionel J. Bovetto, Daniel P. Backer,Jaques R. Villette, Philippe J. Sicard, and Stephane J-L. Bouquelet,Biotechnology and Applied Biochemstry, 15, 48-58, 1992; ShinskeFujiwara, Hirofumi Kakihara, Kim Myung Woo, Andre Lejeune, MitsuhideKanemoto, Keiji Sakaguchi, and Tadayuki Imanaka, Applied andenvironmental microbiology, 58 (12), 4016-4025, 1992; Florian Binder,Otto Huber and August Bock, Gene, 47, 269-277, 1986; Keiji Kainuma,Toshiya Takano and Kunio Yamane, Appl. Microbiol. Biotechnol., 26,149-153, 1987; Takahiro Kaneko, Tetsuo Hamamoto and Koki Horikoshi, J.general Microbiology, 134, 97-105, 1988; Murai Makela, Pekka Mattsson,M. Eugenia Schinina, and Timo Korpela, Biotechnology and Appliedbiochemistry, 10, 414-427, 1988; Ernest K. C. Yu, Hiroyuki Aoki, andMasanaru Misawa, Appl. Microbiol. Biotechnol., 28, 377-379, 1988).

Based on the type of CD principally synthesized, CGTase is classified asα-CGTase, β-CGTase or γ-CGTase. Most of what has been reported in thepast has related to α-, or β-CGTase. Few enzymes have been reported asbeing γ-CGTase (Shigeharu Mori, Susumu Hirose, Takaichi Oya, and SumioKitahata, Oyo Toshitsu Kagaku, 41 (2), 245-253, 1994; Yoshito Fujita,Hitoshi Tsubouchi, Yukio Inagi, Keiji Tomita, Akira Ozaki, and KazuhiroNakanishi, J. Fermentation and Bioengineering, 70 (3), 150-154, 1990;and Takashi Kato and Koki Horikoshi, J. Jpn. Soc. Starch Sci., 33 (2),137-143, 1986).

Enzymes reported to be γ-CGTase is not industrially available becausethe quantity of γ-CD produced is not more than 5 percent, the rate ofproduction of β-CD accelerates in the later stages of the reaction andthus the amount of β-CD produced is equal to or greater than the amountof γ-CD produced, or the amount of γ-CD produced drops precipitously ata substrate concentration of 10 percent or greater and, as acountermeasure, ethanol must be present together in the reactionsolution.

On the other hand, attempts have also been made to modify the structuralgenes of α- or β-CGTase to improve the quantity of γ-CD produced (AkiraNakamura, Keiko Haga, and Kunio Yamane, Biochemstry, 32, 6624-6631,1993; and Michio Kubota, Yoshiki Matsuura, Shuzo Sakai and YukiteruKutsume, Oyo Toshitsu Kagaku, 41 (2), 245-253, 1994). However, these arealso inadequate from an industrial perspective, because even when thequantity of γ-CD produced is increased, the β-CD produced by theoriginal activity is not decreased substantially.

Thus, although α-CD and β-CD are employed in various fields, γ-CD iscurrently little employed. The same is true for CD-containing syrups. CDsyrups comprising principal components in the form of α-CD or β-CD areemployed in various fields, while CD syrup comprising γ-CD as principalcomponent are seldom employed.

BRIEF SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide a newγ-CGTase capable of predominantly producing γ-CD. A further object ofthe present invention is to provide a new microorganism having theability to produce a γ-CGTase for that purpose.

A still further object of the present invention is to provide a methodof manufacturing γ-CD employing the above-mentioned γ-CGTase.

The present inventors conducted a broad search of the natural world formicroorganisms having the ability to produce CGTase producing γ-CD. As aresult, they discovered the desired strain of bacteria having theability to produce CGTase among bacteria belonging to the speciesBacillus clarkii. After culturing this microorganism, producing CGTasein the cultured product, and collecting and identifying the product, itwas discovered that this CGTase is a new enzyme, resulting in achievingthe present invention. It was further discovered that this CGTase couldbe used to predominantly manufacture γ-CD, and an industrial method ofmanufacturing γ-CD was devised.

The present invention relates to cyclodextrin glucanotransferase havingthe enzymatic chemical properties listed below:

(1) Function and substrate specificity: Functioning on starch, dextrin,amylopectin or amylose to produce primarily γ-cyclodextrin, with thequantities of β- and α-cyclodextrin produced being smaller than thequantity of γ-cyclodextrin produced;

(2) Optimum pH: 10.5-11.0;

(3) Optimum temperature: Around 60° C.;

(4) Stable pH: 6-11;

(5) Temperature stability: With a 15 minute-treatment at 50° C.,residual activity of not less than 90 percent is exhibited.

Further, the method of manufacturing cyclodextrin glucanotransferase ofthe present invention is characterized in that a microorganism belongingto the species Bacillus clarkii and having the ability to producecyclodextrin glucanotransferase is cultured, cyclodextringlucanotransferase is produced in the cultured product, and then thecyclodextrin glucanotransferase that has been produced is collected.

In the above-stated method, Bacillus clarkii strain 7364 (FERM BP-7156)may be employed as the microorganism belonging to the species Bacillusclarkii and having the ability to produce cyclodextringlucanotransferase.

Further, the method of manufacturing cyclodextrin of the presentinvention is characterized in that cyclodextrin glucanotransferaseproduced by the species Bacillus clarkii is reacted with a solutioncomprising at least one member selected from among the group consistingof starch, dextrin, aminopectin, and amylose to produce primarilyγ-cyclodextrin and the γ-cyclodextrin produced is collected.

In the method of manufacturing cyclodextrin of the present invention setforth above, the cyclodextrin glucanotransferase can be the cyclodextringlucanotransferase of the present invention.

The present invention further relates to Bacillus clarkii strain 7364(FERM BP-7156), which is a bacteria of Bacillus clarki species havingthe ability to produce cyclodextrin glucanotransferase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of pH on the activity of γ-CGTasederived from Bacillus clarkii 7364 (Blue value method).

FIG. 2 is a graph showing the effect of temperature on the activity ofγ-CGTase derived from Bacillus clarkii 7364 (Blue value method).

FIG. 3 is a graph showing change over time in the γ-CD productionreaction using soluble starch as a substrate.

DETAILED DESCRIPTION OF THE INVENTION

(Cyclodextrin Glucanotransferase)

Table 1 gives the bacteriological properties of the strain newlydiscovered and separated by the present inventors.

TABLE 1 Various properties of the bacterial strain Culture temperature37° C. Cell form Rod-shaped microorganism (0.6-0.8 × 3-5 μm) withelongated form Gram dyeing + Spores ± Motility + Colony form Shape ofcolony: irregular Perimeter of colony: filamentous Colony surfaceprotrusions: low and flat Luster: None Color: Cream Size: Φ 3-4 mmCharacteristics: Colonies adhere to medium Catalase + Oxidase + O/F Test− β-Galactosidase + Arginine hydrolase − Lysine decarboxylase −Ornithine decarboxylase − Use of citric acid − H₂S production − Urease −Tryptophan deamidase − Indole production − Acetoin production +Gelatinase − Nitrate reduction − Hydrolysis Casein + Gelatin + Starch +Tween 20 − Tween 40 + Tween 60 + Phenyl alanine deamidase − Growthproperties 10° C. − 40° C. + 50° C. − pH 7 −  5% NaCl + 10% NaCl +Identification result Bacillus clarkii

The 16 S rDNA nucleotide sequence of this bacterial strain is shown asSEQ ID NO:1 in the sequence listing.

This bacterial strain is a motile gram-positive rod-shaped microorganismalso exhibiting an extended form. It is positive for both catalase andoxidase. Although determination of the form of the spores was difficult,the impression was obtained of terminal spores on extended formextremities, and it was presumed to belong to the genus Bacillus. Inaddition, bacterial body fatty acid composition (CFA), 16 S rDNAnucleotide sequencing, and tests of utilization of saccharides wereconducted. Since the fastest proliferation rate was achieved with thisbacterial strain in the alkaline range and it would not grow at pH 7.0or below, it was categorized into alkalophilic bacteria. Although it hada low homology ratio of 96.12 percent in 16 S rDNA nucleotide sequencingwith previously reported Alkalophilic Bacillus clarkii (Preben Nielsen,Dagmar Fritze and Fergus G. Priest, Microbiology, 141, 1745-1761, 1995),it was presumed to be a related species. However, in other physiologicalproperty tests, it exhibited a good match with values reported by I.Yumoto et al. (I. Yumoto et al., Int. J. Syat. Bacteriol., 48, 565-571,1998), and this bacterial strain was presumed to be Bacillus clarkii.

These results clearly show this bacterial strain to be a new bacterialspecies, which has been named Bacillus clarkii strain 7364. Thisbacterial strain has been deposited with Kogyo Gijutsuin Seimei KogakuKogyo Gijutsu Kenkyujo (currently, the Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology, 1-1, 1chome, Tsukuba Higashi, Ibaraki Prefecture, Japan) as FERM BP-7156.

To date, no other bacterial strain of Bacillus clarkii having theability to produce CGTase has been reported within Bacillus clarkiispecies. Further, the related species of B. horti (JCM No. 9943T), B.clarkii (ATCC No. 700162), and B. agaradhaerens (ATCC No. 700163) havebeen confirmed not to have CGTase activity. That is, the presentbacterial strain is the first bacterial strain belonging to the Bacillusclarkii species to have the ability to produce CGTase.

(Method of Manufacturing Cyclodextrin Glucanotransferase)

To manufacture CGTase using Bacillus clarkii strain 7364, themicroorganism is vigorously grown and then cultured in a synthetic ornatural medium comprising the carbon source, nitrogen source, inorganicsalts, necessary nutrients, and the like that are necessary for thesmooth production of the enzyme. Examples of carbon sources suitable foruse are carbohydrates such as starches and compositional fractionsthereof, baked dextrin, processed starches, starch derivatives,physically processed starches, and α-starches. Specific examples aresoluble starch, corn starch, potato starch, sweet potato starch,dextrin, amylopectin, and amylose. Examples of nitrogen sources areorganic nitrogen source substances such as polypeptones, casein, meatextract, yeast extract, corn steep liquor, soybeans, soybean cakes, andother extracts; inorganic nitrogen compounds such as ammonium sulfateand ammonium phosphate; and amino acids such as glutamic acid. Examplesof inorganic salts suitable for use are phosphates such as monopotassiumphosphate and dipotassium phosphate; magnesium salts such as magnesiumsulfate; calcium salts such as calcium chloride; and sodium salts suchas sodium carbonate. Culturing is desirably conducted under aerobicconditions by shake culturing or stir culturing with ventilation in amedium adjusted to greater than pH 7, preferably to within the range ofpH 8 to 11, at a temperature falling within the range of 10-45° C.,preferably 30-42° C. However, culturing is not specifically limitedthereto and may be conducted under other conditions so long as themicroorganism grows and the targeted enzyme is produced.

When cultured under the conditions as mentioned above, a substantialamount of CGTase is normally produced in the culture solution about 48hours after the start of culturing. Next, the bacterial body is removedfrom the culture solution, yielding a filtered culture solution. This isdesalted with an ultrafiltration membrane and concentrated, and theenzyme is recovered. The roughly purified enzyme thus obtained may beused as it is in the CD production reaction. However, as necessary, itcan be employed following purification by ammonium sulfate salting out;precipitation from an organic solvent; adsorption elution byDEAE-Sephadex or butyl Toyopal; column fractionation by Sephadex,Toyopal and the like; and affinity chromatography in which γ-CD isderived as a ligand.

Methods of measuring enzyme activity are described below.

(CGTase Activity Measurement)

CGTase activity was measured at 40° or 50° C. using 50 mMglycine-NaCl—NaOH buffer solution (pH 10.0).

(Blue Value Method)

50 mg of amylose (EX-III type made by Hayashibara) was dissolved in 2 mLof 1 N NaOH overnight, neutralized with 1 N HCl, added 50 mMglycine-NaCl—NaOH buffer (pH 10.0) to 50 mL, yielding a substratesolution. A 300 μL of the substrate solution was maintained at atemperature of 40° C. for 10 minutes, 200 μL of a suitably dilutedenzyme solution was added to start the reaction, and the reaction wasconducted for 10 minutes at the same temperature. The reaction was thenstopped by adding 4 mL of 0.2 N HCl. To the reaction solution, 4 mL ofwater and 500 μL of 0.02 percent I2-0.2 percent KI solution were added.The absorbance at 700 nm was measured. A control was prepared bysimilarly adding 0.2 N HCl and then adding an enzyme solution. One unitwas defined as the quantity of enzyme reducing the absorbance at 700 nmunder these conditions by 10 percent in one min. relative to thecontrol.

(γ-CD Production Activity)

One mL of soluble starch (nacalai tesque) was dissolved in 25 mMglycine-NaCl—NaOH buffer (pH 10.0) to 10% (w/v) and heated at 50° C. for10 minutes. A 200 μL of a suitably diluted enzyme solution was added tostart the reaction, the reaction was continued for 30 minutes at thesame temperature, and the reaction was stopped by adding 3 mL of 0.02 NHCl. A 20 μL of the reaction solution was subjected to HPLC and thequantity of γ-CD produced was measured. One unit was defined as theamount of enzyme producing 1 μmol/mL of γ-CD per minute under theseconditions.

The HPLC conditions employed in this measurement are as follows:

Column YMC-pack AQ-312 (6 × 150 mm) Solvent 10 percent MeOH Flow rate1.0 mL/min Temp. R. T. Detection RI ATT 7

A CD solution of prescribed concentration was analyzed under theabove-stated conditions and a calibration curve having the followingequation was obtained by calculating the correspondence between the CDcontent and the HPLC area.(HPLC area)=3.092×106×(γ-CD w/v %) R2=0.999(Analysis of Quantity of CD Produced)

The CD content in the reaction solution was analyzed by HPLC. Theanalysis conditions are as follows.

HPLC Method 1 Column Aminex HPX-42A (Bio-Rad Lab. 7.8 × 300 mm) SolventH₂O Flow rate 0.5 mL/min Temp. 55° C. Detection RI ATT 9

HPLC Method 2 was identical to the method employed to analyze CDproduction activity.

The CD production reaction was conducted using soluble potato starch(Sigma) as a substrate.

The various enzymological properties of the CGTase obtained are givenbelow.

A 200 μL of the enzyme (100 μg of protein) and 1.0 percent (w/v) amylose(DP 117) dissolved in various buffers of pH 3-11.9 were reacted for 10min at 40° C. and the relative activity (Blue value method) wascalculated. Further, 50 μL of enzyme solution (25 μg of protein) and 500μL of various buffers with a pH ranging from 3 to 11.9 were combined andthen left standing at 4° C. for 24 hours. After adding 2.75 mL of 50 mMglycine-NaOH buffer (pH 10.0), the residual activity was measured (FIG.1). The results revealed that the optimum pH of the enzyme and the pHstability range were 10.5-11.0 and 6-11.0, respectively.

A 200 μL of the enzyme (100 μg of protein) and 1.0 percent (w/v) amylose(DP 117) dissolved in 50 mM glycine-NaOH buffer (pH 10.0) were reactedfor 10 min at various temperatures and the relative activity (Blue valuemethod) was calculated. Further, 20 μL of enzyme solution and 1,180 mLof 50 mM glycine-NaOH buffer (pH 10.0) were mixed and then left standingat various temperatures for 15 min. The residual activity was measured(FIG. 2). The results revealed that the optimum temperature of theenzyme and the temperature stability range were 60° C. and 30° C. orbelow, respectively. These results were identical for γ-CD productionactivity.

A comparison of the various physical properties of the enzyme in theform of the above results, the molecular weight of the enzyme, andisoelectric point examination results are compared in Table 2 to thoseof CGTase derived from other bacterial strains.

TABLE 2 A comparison of various physical properties with CGTase of othersources already reported Bacterial B. Clarkii B. sp. B. megaterium B.circulans B. macerans strain 7364 strain AL-6 B. stearothermophilus (F1F2) (F1 F2) (IFO 3490) Isoelectric point 3.98 — 4.45 6.07 6.80 5.80 6.604.60 Optimum pH 10.5-11.0  7.5-10.5 6.0  5.2-6.2  5.2-5.7 5.2-5.7 Optimum temp. 60°C. 55° C. 70° C. 55° C. 55° C. 55° C. Stability (pH) 6.0-11.0 5-8 7.0-9.2 7.0-10.0 7.0-9.0 8.0-10.0 Stability (temp) 30° C.40° C. 50° C. 55° C. 55° C. 55° C. Molecular 66,000 74,000 68,000 66,000— 65,000 weight Main product γ γ, β α, β β β α

The molecular weight of CDTase derived from Bacillus Clarkii 7364 strainwas 66,000 by SDS-PAGE and the isoelectric point thereof was 3.98(focusing electrophoresis).

(Method of Manufacturing Cyclodextrin)

CD can be manufactured with the CDTase of the present invention, forexample, by adding 0.5-300 U (per gram of dry starch) of the presentenzyme solution (purified enzyme or crude enzyme) to an aqueous solutioncomprising 1-30 percent starch (including starch or a compositionalfraction thereof, dextrin, amylopectin, amylose, or some other processedstarch) and conducting an enzymatic reaction for 1-96 hours at 20-60° C.from pH 4.5-12. As needed, the starch can be preheated or subjected to aliquefaction treatment for use. More γ-CD than α-CD and β-CD iscontained in the sugars (syrup) prepared by the method as mentionedabove. In addition to these CDs, monosaccharides such as glucose andvarious oligosaccharides (maltose and the like), dextrin, and the likeare sometimes contained. Further, as needed, a CD having a desiredsingle degree of polymerization can be separated (by crystallization,chromatofractionation, fermentation by yeast or the like, enzymictreatment, or the like) for use. That is, γ-CD can be separated andpurified from the above-described product. Since the reaction solutionobtained by the method of the present invention comprises more γ-CD thanα-CD and β-CD, the γ-CD is more readily separated and purified than inprior art. In addition to the syrup obtained by the above-mentionedmethod, the form may be any other form such as crystals, freeze-driedproduct, powder, or granules.

The CD (particularly CD with a high γ-CD content or highly purifiedγ-CD) manufactured by the manufacturing method of the present inventioncan be employed in all food and drink products suited to oralconsumption in the same manner as currently available CD. Examples ofsuch food and drink products are beverages such as tea and refreshmentbeverages; Japanese and Western snacks such as candies, jellies, andJapanese finger foods; milk products such as yogurt and ice-cream;processed meat products such as ham and sausage; processed seafoodproducts such as boiled fish paste and Naruto; noodles; pickledvegetables; other prepared foods; and instant foods. Further, the CDmanufactured by the manufacturing method of the present invention can beadded and incorporated to stabilize or emulsify fragrance materials andthe like, or as an excipient or the like, to conveniently andeffectively enhance the desirability and functionality thereof. Further,in addition to foods and beverages, the CD manufactured by themanufacturing method of the present invention can be employed tostabilize the active ingredients of, and emulsify, not only foods anddrinks, but also pharmaceuticals, cosmetic products and the like, and asan excipient.

The method of use of the CD manufactured by the manufacturing method ofthe present invention is not specifically limited so long as the CD ispresent together in a food or drink product, pharmaceutical, orcosmetic. For example, the CD can be added simultaneously with theprocessing of the food or drink material serving as the base or addedafter completion of processing of the base food or drink product. Itsuffices to employ a method of addition suited to the actual conditionsof the process of manufacturing the individual food product.

In the case of food and drink products, the quantity of CD added is notspecifically limited so long as the base food or drink product does notlose its original flavor or aroma. Generally, a quantity added of notgreater than 20 weight percent is desirable. Equal to or greater than 20weight percent is undesirable because it is apprehended that thedesirability is diminished by changing the flavor or aroma of the basefood or drink product by the masking effect of the CD. In the case ofpharmaceuticals and cosmetics, there is no particular limitation so longas the effect of the active ingredients is not lost; the use of not morethan 50 weight percent is desirable.

The inventions disclosed in the present specification relate toinventions described in Japanese Patent Application No. 2000-151053filed with the Japan Patent Office on May 23, 2000. The full disclosureof the Japanese patent application is expressly incorporated herein byreference.

EMBODIMENTS

The present inventions are more specifically described below throughembodiments.

Embodiment 1

Bacillus clarkii 7364 strain (FERM BP-7156) was cultivated with shakingfor 48 hours at 37° C. in a liquid medium comprising a carbon source inthe form of 1.0 percent (w/v) of Neotack #30T (made by Nihon ShokuhinKako Co., Ltd.), a nitrogen source in the form of 0.5 percentSoyflower-FT (made by Nisshin Oil Mills, Ltd.), 0.5 percent yeastextract (made by Difco), 0.1 percent K₂HPO₄, 0.02 percent MgSO₄.7H₂O,and 0.8 percent Na₂CO₃. CGTase was secreted into the culture solution(Blue value method 20 U/mL culture supernatant).

The CGTase obtained was purified by affinity chromatography. Thephysical properties thereof are given in Table 2.

Embodiment 2

The culture supernatant solution obtained in Embodiment 1 wasconcentrated with a UF concentration membrane (PM-10) to obtain a crudeenzyme solution and a saccharification test was conducted. Theconcentrated solution had an activity of 55 U/mL (Blue value method). Asubstrate solution was first prepared by dissolving a substrate in theform of soluble starch to 10 percent (w/v) in 50 mM glycine-NaCl—NaOHbuffer (pH 10.0). The crude enzyme solution was then added in a manneryielding 80, 40, 20, 10, and 5 U/g-DS to 5 mL of the substrate solutionand reacted at 50° C. Sampling was conducted at hours 2, 4, 6, 24, and48 from the start of the reaction. HPLC Methods 1 and 2 were thenemployed to calculate the quantities of γ-CD produced (FIG. 3). Theresults revealed that when 80 U/g of DS was added, produced were 9.7percent γ-CD, 1.7 percent α-CD, and 0.9 percent β-CD (HPLC area) in 48hours .

Embodiment 3

Corn starch was liquefied by the usual method using α-amylase to preparea liquid starch solution with a concentration of 20 weight percent and aglucose equivalent of 7. Next, the liquid starch solution was adjustedto pH 7. The culture supernatant solution described in Embodiment 1 wasconcentrated using an UF concentration membrane (PM-10) to obtain acrude enzyme solution, to which added 450 unit/g substrate and reactedfor 48 hours at 55° C. Subsequently, the reaction solution was heated todeactivate the enzyme. Purification such as decoloration andion-exchange were conducted to prepare a CD-comprising syrup. The sugarcomposition thereof is given in Table 3. The CD content was determinedby HPLC Methods 1 and 2.

TABLE 3 α-CD 1.7 percent β-CD 0.8 percent γ-CD 8.2 percent Other 89.3percent  sugarsEmbodiment 4

The reaction solution of Embodiment 2 (a reaction solution to which 80U/g of DS was added and reacted for 48 hours) was processed withglucoamylase, placed on an activated carbon column (φ2.5×20 cm), andwashed with pure water to remove the glucose produced by glucoamylaseprocessing. After further washing with 20 percent ethanol, adsorptionfractions were eluted out with a threefold quantity of 25 percentethanol. The eluted fractions were concentrated and then cooled for 15min at 4° C. to precipitate β-CD, which was removed by centrifugation.Next, the supernatant was placed on Toyopal HW-40S (φ2.9×90 cm). Whenthe Brix of each 10 mL fraction was measured, a single principal peakwas observed (Tube: 23-28). Each fraction was analyzed by HPLC,revealing that fractions 23-26 were either α-CD or mixtures of α-CD andγ-CD. Thus, fractions 27 and 28 were collected, concentrated, and freezedried. Determination by HPLC revealed that these fractions were γ-CDwith a purity of 99.5 percent. Finally, 175 mg of γ-CD was isolated.This γ-CD was confirmed to be γ-CD by NMR.

¹³C-NMR data: 104.30, 83.08, 75.56, 74.93, 74.40, 62.86 ppm.

INDUSTRIAL APPLICABILITY

The present invention provides a microorganism having the ability toproduce a new γ-CGTase and a new γ-CGTase capable of predominantlyproducing γ-CD. The present invention further provides a method ofefficiently manufacturing γ-CD using γ-CGTase.

1. Cyclodextrin glucanotransferase having the enzymatic chemicalproperties listed below: a) function and substrate specificity:enzymatically acting on starch, dextrin, amylopectin, and amylose toproduce primarily γ-cyclodextrin, with the quantities of β- andα-cyclodextrin produced being smaller than the quantity ofγ-cyclodextrin produced; b) optimum pH: 10.5-11.0; c) optimumtemperature: about 60° C.; d) stable pH: 6-11; e) temperature stability:with a 15 minute-treatment at 50° C., residual activity of not less than90 percent is exhibited.
 2. A method of manufacturing cyclodextringlucanotransferase wherein a microorganism belonging to the speciesBacillus clarkii that produces cyclodextrin glucanotransferase iscultured to produce cyclodextrin glucanotransferase, and thecyclodextrin glucanotransferase that has been produced is collected. 3.The manufacturing method according to claim 2, wherein the microorganismbelonging to the species Bacillus clarkii and having ability to producecyclodextrin glucanotransferase is Bacillus clarkii strain 7364 (FERMBP-7156).
 4. A method of manufacturing cyclodextrin wherein cyclodextringlucanotransferase produced by the species Bacillus clarkii is reactedwith a solution comprising at least one member selected from the groupconsisting of starch, dextrin, amylopectin, and amylose to produceprincipally γ-cyclodextrin.
 5. The manufacturing method according toclaim 4, wherein the cyclodextrin glucanotransferase having theenzymatic chemical properties listed below: a) function and substratespecificity: enzymatically acting on starch, dextrin, amylopectin, andamylose to produce primarily γ-cyclodextrin, with the quantities of β-and α-cyclodextrin produced being smaller than the quantity ofγ-cyclodextrin produced; b) optimum pH: 10.5-11.0; c) optimumtemperature: about 60° C.; d) stable pH: 6-11; e) temperature stability:with a 15 minute-treatment at 50° C., residual activity of not less than90 percent is exhibited.
 6. The manufacturing method according to claim4, wherein the γ-cyclodextrin produced is separated from othercyclodextrins.
 7. Bacillus clarkii strain 7364 (FERM BP-7156) being thespecies Bacillus clarkii having the ability to produce cyclodextringlucanotransferase.